The ability to detect mutations in coding and non-coding DNA, as well as RNA, is important for the diagnosis of inherited disease. A gene mutation can be a single nucleotide change or multiple nucleotide changes in a DNA sequence encoding an essential protein. A single nucleotide change or multiple nucleotide changes can result in a frame shift, a stop codon, or a non-conservative amino acid substitution in a gene, each of which can independently render the encoded protein inactive. However, a gene mutation can be harmless, resulting in a protein product with no detectable change in function (i.e. a harmless gene polymorphism). Mutation in repetitive DNA can also lead to disease, as in, for example, human fragile-X syndrome, spinal and bulbar muscular dystrophy, and myotonic dystrophy.
A mutant nucleic acid which includes a single nucleotide change or multiple nucleotide changes will form one or more base pair mismatches after denaturation and subsequent annealing with the corresponding wild type and complementary nucleic acid. For example, G:A, C:T, C:C, G:G, A:A, T:T, C:A, and G:T represent the eight possible single base pair mismatches which can be found in a nucleic acid heteroduplex, wherein U is substituted for T when a nucleic acid strand is RNA. Nucleic acid loops can form when at least one strand of a heteroduplex includes a deletion, substitution, insertion, transposition, or inversion of DNA or RNA. Several screening methods have been designed to detect DNA mismatches in a heteroduplex. These methods include RNAse A digestion, chemical cleavage, as well as PCR- and primer extension-based detection methods (reviewed in Cotton, Curr. Opinion in Biotech. 3, 24 (1992)).
The resolvases (e.g. X-solvases of yeast and bacteriophage T4, Jensch et al. EMBO J. 8, 4325 (1989)) are nucleolytic enzymes capable of catalyzing the resolution of branched DNA intermediates (e.g., DNA cruciforms) which can involve hundreds of nucleotides. In general, these enzymes are active close to the site of DNA distortion (Bhattacharyya et al., J. Mol. Biol., 221, 1191, (1991)). T4 Endonuclease VII, the product of gene 49 of bacteriophage T4 (Kleff et al., The EMBO J. 7, 1527, (1988)) is a resolvase (West, Annu. Rev. Biochem. 61, 603, (1992)) which was first shown to resolve Holliday-structures (Mizuuchi et al., Cell 29, 357, (1982)). T4 Endonuclease VII has been shown to recognize DNA cruciforms (Bhattacharyya et al., supra; Mizuuchi et al., supra) and DNA loops (Kleff et al., supra), and it may be involved in patch repair. Bacteriophage T7 Endonuclease I has also been shown to recognize and cleave DNA cruciforms (West, Ann. Rev. Biochem. 61, 603, (1992)). Eukaryotic resolvases, particularly from the yeast Saccharomyces cerevisiae, have been shown to recognize and cleave cruciform DNA (West, supra; Jensch, et al., EMBO J. 8, 4325 (1989)).
Other nucleases are known which recognize and cleave DNA mismatches. For example, S1 nuclease is capable of recognizing and cleaving DNA mismatches formed when a test DNA and a control DNA are annealed to form a heteroduplex (Shenk et al., Proc. Natl. Acad. Sci. 72, 989, (1975)). However, the rate of cleavage is unacceptably slow (Dodgson et al., Biochemistry 16, 2374, (1977)). The Nut Y repair protein of E. coli is also capable of detecting and cleaving DNA mismatches. However, the Mut Y repair protein is only capable of detecting 50% of the total number of mutations occurring in a mutant DNA segment (Lu et al., Genomics 14, 249, (1992)).